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Aquatic Toxicology 94 (2009) 4046

Contents lists available at ScienceDirect

Aquatic Toxicology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a q u a t o x

How aluminium exposure promotes osmoregulatory disturbances in theneotropical freshwater fish Prochilus lineatus

Marina M.P. Camargoa,b, Marisa N. Fernandesa, Cludia B.R. Martinez b,

a Departamento de Cincias Fisiolgicas, Universidade Federal de So Carlos, Rod. Washington Luis, Km 235, 13565-905 So Carlos, So Paulo, Brazilb Departamento de Cincias Fisiolgicas, Universidade Estadual de Londrina, Rod. Celso Garcia Cid (PR-445), Km 374, 86051-990 Londrina, Paran, Brazil

a r t i c l e i n f o

Article history:

Received 3 April 2009Received in revised form 4 May 2009

Accepted 27 May 2009

Keywords:

Chloride cellsGills

Metal

Na+/K+ATPase

Stress response

a b s t r a c t

The aim of this study was to understand the effects of the interaction between aluminium and low pH

in a native fish species Prochilodus lineatus. Thus, juveniles of this neotropical fish species were exposedto 196g L1 of dissolved aluminium in acid water (Al group), only to acid water (pH group) or to waterwith neutral pH (CTR group) for 6, 24 and 96 h. Al effects were evaluated with regard to hematological

parameters (hemoglobin, hematocrit and red blood cell number), plasma ions and osmolarity, densityand distributionof chloride cells (CC), Na+/K+ATPaseactivity in the gills,metabolic (proteinand glucose)

and endocrine (cortisol) parameters. The fish exposed to Al had increased hematological and metabolicparameters in relation to the CTR group after all periods of exposure. Infish exposedto Al for 24and 96h

plasmaions andosmolarity were significantlylower and theidentification of theenzyme Na+/K+ATPaseby immunohistochemistry indicated a reduction in the number of CC in the gills. Enzyme activity was

50%lower in fish exposed to Al in all experimental times. Taken together these results showedthat acuteexposure to Al causes an ionic unbalance, probably related to the effects of Al on Na+/K+ATPase activity,

on the distribution and number of chloride cells in the gills as well as the effects associated with thestress response caused by the presence of the metal.

2009 Elsevier B.V. All rights reserved.

1. Introduction

Aluminium is the most abundant metal on earth and mostlyoccurs as oxide and silicate of aluminium (Scancar et al., 2004).Aluminium is also found in the atmospheric air of thebig cities and

industrialized areas (Casarini et al., 2001), and is used as a floc-culation agent in water treatment (Silva et al., 2007). Some areasof England, United States and Czech Republic have high concentra-tions ofAl in their river waters, reaching up to1350g L1 of totalAldue to air pollution and acid rain (Guibaud and Gualthier, 2003). InBrazil, thismetalis naturally found in theAmazon regionsoil wherethe water of the rivers and streams has naturally low pH (Hara and

Oliveira, 2004; Artaxo et al., 2005; Horbe et al., 2005). In the stateof So Paulo (SoutheastBrazil), 35% of the examined surface watersdestined for public consumption contain high levels of dissolved Al(1005700g L1) (CETESB, 2008). Even though, according to theBrazilian law, the limits for dissolved Al in freshwater is between

100 and 200g L1 (CONAMA 357, 2005).Aluminium is toxic to fishes and most studies on Al toxicity are

restricted tofish species from theNorthernhemisphere(McCartney

Corresponding author. Tel.: +55 43 3371 4650; fax: +55 43 3371 4467.

E-mail address: [email protected](C.B.R. Martinez).

et al., 2003; Monette and McCormick, 2008). In the tropical and

neotropical areassuch studiesare still rare (Barcarolli andMartinez,2004), leaving a gap in the knowledge of the physiological effectsof Al in neotropical fishes where the high temperature of naturalwater may increase toxicity (Lydersen et al., 1990).

Most of the studies on Al toxicity in fish are related to the pH ofwater, as the solubility of Al increases linearly with the reductionin pH increasing the presence of inorganic Al, the form of Al mosttoxic to fish (Gensemer and Playle, 1999). In contrast, acidity, by

itself, causes several effects in fishes such as hematological (Woodand McDonald, 1982; Carvalho and Fernandes, 2006), endocrineand metabolic (Cole et al., 2001) and reproductive (Vuorinen et al.,

2003) disturbances. However, when acidity is associated with Al inwater the effects are concentrated mainly in the gills and all phys-iological processes related to this organ (Waring and Brown, 1995;Cole et al., 2001; Teien et al., 2006). The gills are a multi-functionalorgan playing an important role in osmoregulation of fish (Hwang

and Lee, 2008). This organ represents the main target-organ of pol-lutants due to its extensive surface area in contact with the externalenvironment and the very thin barrier between the environmentalwater and internal milieu of fish (Dang et al., 2000; Cerqueira and

Fernandes, 2002). Gills are the most affected organ by Al contami-nated water (Dietrich and Schlatter, 1989; Playle and Wood, 1990;Peuranen et al., 1993).

0166-445X/$ see front matter 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquatox.2009.05.017
http://www.sciencedirect.com/science/journal/0166445Xhttp://www.elsevier.com/locate/aquatoxmailto:[email protected]://dx.doi.org/10.1016/j.aquatox.2009.05.017http://dx.doi.org/10.1016/j.aquatox.2009.05.017mailto:[email protected]://www.elsevier.com/locate/aquatoxhttp://www.sciencedirect.com/science/journal/0166445X


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M.M.P. Camargo et al. / Aquatic Toxicology 94 (2009) 4046 41

In the gills the main cells related to ionic regulation are the chlo-ride cells (Hirose et al., 2003), which are located mostly in the gill

filaments,closeto thebase of thelamellae(Perry, 1997; Hirose et al.,2003). Chloride cells (CC) are large andround cells characterized bynumerous mitochondria and an extensive tubular membrane sys-tem containing a high density of Na+/K+ATPase activity (Dang et

al., 2000).In order to maintain their body fluid and mineral homeostasis,

freshwater teleosts compensate for diffusive ion loss and osmoticgain of water by actively absorbing Na+, CI and Ca2+ through the

gills and producing large volumes of diluted urine, respectively(Hirose et al., 2003). Thus, ion analysis and plasma osmolarity,associated with the determination of density and localization ofCC, besides Na+/K+ATPase activity, which actively transport ions

through the gills, should be informative for the understanding ofthe mechanism of Al toxicity in freshwater teleosts (Peuranen etal., 1993; Vuorinen et al., 2003). Therefore, in the present studyan integrated approach examining plasma ions and osmolarity, CC

distribution and density, gill Na+/K+ATPase activity, and hemato-logical parameters besides others associated with stress response,was employed in order to evaluate the effect of acute exposureto aluminium in acid pH on the osmoregulation of the Prochilo-

dus lineatus fish. This species was chosen because it represents

a neotropical fish commonly found in rivers of the south andsoutheast regions of Brazil, and it is also considered a potentialbioindicator species (Martinez et al., 2004; Takasusuki et al., 2004;

Simonato et al., 2006).

2. Materials and methods

Juveniles P. lineatus (Valenciennes, 1847) (n = 115) weighing20.076.08 g and total length equals 12.231.23cm (mean SD)were obtained from the hatchery station of State University of Lon-

drina. Prior to experiments, the fish were acclimated, for 7 days,in 300 L tanks with non-chlorinated water, constant aeration and aphotoperiod of 12h:12h. During acclimation, the animals were fed

with commercial pellet food with 36% protein (Guabi, BR) every2 days, and the feeding was suspended 24 h before the beginningof the toxicity tests. The physical and chemical parameters of thewater were continuously monitored (T= 21.80.9 C; pH 7.50.1;DO=7.50.7mgO2 L

1; conductivity= 133.49.7S cm1; hard-ness=42.56.0mgCaCO3 L

1).After acclimation, groups of fish (6 or 7 per aquarium) were

transferred to glass aquaria (100L each) containing water as fol-lows: fish of the control group (CTR) to water with neutral pH; fish

of pH group (pH) to water with acid pH (5.0); fish of Al group (Al) towater in acid pH (5.0) + aluminium. Acid pH in water was obtainedby the addition of 50% HCl and the aluminium was added to waterasAl2(SO4)3. The toxicity tests in each experimental time(6, 24 and

96 h) were performed in separated aquaria. All toxicity tests were

carried out in duplicate.During the tests, water was monitored for temperature, pH,

dissolved oxygen and conductivity. Samples of water collected

immediately after each experimental period were analysed for Alconcentration, using atomic absorption spectrophotometry. Theconcentration of total Al was determinedin samples of non-filteredwater and the concentration of dissolved Al was determined in fil-

tered water samples (0.45m); for both analyses, samples wereacidified with HNO3.

At the end of each experimental period, fish were anaesthetisedwith benzocaine (0.1 g L1) and a blood sample was withdrawn, via

the caudal vein, using heparinised plastic syringes. The animalswere then killed by medullar section and the gills were removedand processed for immunohistochemical against Na+/K+ATPase

and enzyme assay. Immediately after sampling, the blood was cen-

trifuged (10 min, 10,000g) and samples of plasma were frozen(20 C) for osmolarity, ion concentrations, cortisol, glucose and

protein analyses.

2.1. Hematological analysis

Blood samples were used to determine the hematocrit (Hct),hemoglobin content (Hb) and red blood cells count (RBC). Hctwas determined by micro-hematocrit technique, using heparinised

capillary tubes and centrifuged for 5 min in an appropriate cen-trifuge. Hemoglobin wasdeterminedby the cyanometahemoglobinmethod, in a spectrophotometer (Libra S32, Biochrom, UK) using acommercial kit (Analysa, Brazil). Red blood cells were counted in

blood samples fixed in formol-citrate buffer, using an improvedNeubauer chamber under a light microscope (magnification of400X).

2.2. Identification of chloride cells in the gills

The gills were washed with saline solution and samples

from the gill were fixed in Bouins fluid (6 h), dehydrated inethanol crescent series and embedded in paraffin. Sagittal sec-tions (8m in thickness) were made and processed according tothe avidinbiotinperoxidase complex (ABC) technique to visu-

alize chloride cells, through the identification of Na+/K+ATPase,according to the method described by Dang et al. (2000).Slides were incubated with a mouse monoclonal antibody toNa+/K+ATPase (IgG5) and goat-anti-mouse IgG was used asthe second antiserum. Subsequently, 3-3-diaminobenzidine (DAB0.05M) in Tris-buffered saline (pH 7.4), containing H2O2 (0.03%)was applied. Finally, sections were dehydrated and mounted.

The chloride cells were quantified in relation to the filament

length (mm) according to their localization: in the gill filament(CCF) or in the gill lamellae (CCL), using a photomicroscope(DM 2500, Leica, Germany) and an image analyser (Leica Qwin,Germany). For each section from the same fish, five filaments

were randomly selected and measured for CC quantification. The

results were expressed as the number of CC per mm of filament(meanSD).

2.3. Na+/K+ATPase activity in the gills

After washing the gill arches, the gill filaments were removed

andtransferred toplastic tubes containing SEIbuffer (sucrose 0.3M,Na2EDTA 0.1 mM,imidazole 0.03 M,-mercaptoethanol 10mM, pH7.4) and then kept frozen (20 C) until the moment of enzymeassay. For assay, the gill filaments were homogenised with SEI

buffer (10 volume) and centrifuged (10,000g, 15min, 4 C).The supernatant was used to determine Na+/K+ATPase activ-ity, according to the method described by Quabius et al. (1997)and adapted for a microplate reader by Nolan (2000). The assay

consists of determining the phosphate released by the samplesincubated in buffer (NaCl 100 mM, MgCl2 8 mM, imidazole 30mM,

Table 1

Physical and chemical parameters of the water samples collected in the different

groups during the tests (6, 24 and 96 h).

Parameter CTR group pH group Al group

Temperature (C) 22.40.5 22.40.5 22.5 0.5

pH 7.60.5 5.10.3 5.2 0.1

Dissolved oxygen (mgO2 L1) 7.61.0 7.40.9 7.5 0.8

Conductivity (S cm1) 83.723.6 104.118.3 104.6 15.0

Hardness (mgCaCO3 L1) 41.37.9 41.65.9 44.1 7.3

Total Al (g L1) ND ND 438.0 36.3

Dissolved Al (g L1) ND ND 196.0 28.7

The values represent meansSD (n = 5). ND: not detected.


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42 M.M.P. Camargo et al. / Aquatic Toxicology 94 (2009) 4046

Fig. 1. Hemoglobin (A), hematocrit (B) and number of erythrocytes (C) ofP. lineatus

exposed to 6, 24 and 96 h to CTR, pH or Al groups. The bars indicate mean and the

verticallines, the SD (number of animals:1013).*Different fromthe CTR group and#different from the pH group for each experimental period (P< 0.05).

EDTA 0.1 mM, ATP 3mM, pH 7.6) containing KCl (5mM) or ouabain

(2.5mM). A solution of 0.65mM phosphate (Sigma) was used asstandard and the samples were analysed in triplicate at 620 nm ina microplate reader (ELX 800, BioTek, USA). Na+/K+ATPase activ-ity was expressed asmolPi/mgprotein h1. Protein concentrationwas determined according to the method described by Lowry et al.(1951).

2.4. Plasma ions and osmolarity

The concentrations of Na+ and K+ were measured in plasmausing a flame photometer (Analyser, Brazil). The concentration ofCl was determined with the thiocyanate method in spectropho-tometer at 470nm (commercial kit, Analisa, Brazil). Osmolarity

was determined using a freezing point osmometer (Osmomat 030,Gonotec, Germany).

2.5. Plasma concentrations of cortisol, glucose and proteins

Cortisol was determined in plasma with a commercial immu-

noenzymatic assay kit (Diagnostic Systems, Laboratories, USA),andthe absorbance was read in a microplate reader at 450 nm. The

concentration of glucose was determined using a commercial col-orimetric kit (Labstest, Brazil) at 505 nm in a spectrophotometer.

Plasma protein concentration was determined according to themethod described by Lowry et al. (1951), using bovine serum albu-min (BSA) as standard.

2.6. Statistical analysis

The results are presented as meansSD. The results obtained ineach treatment (CTR, pH or Al), at each experimental time (6, 24 or

96 h), were compared using one-way analysis of variance (ANOVA)or KruskallWallis test, depending on datanormal distribution andhomogeneity of variance. Differences were analysed by a post hocTukey test for all pairwise comparisons between treatments. Sta-

tistical significance was designated asP


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Fig. 3. Plasmatic concentrations of proteins (A), glucose (B) and cortisol (C) of P.

lineatus exposed to 6, 24 and 96 h to CTR, pH or Al groups. The bars indicate mean

and the vertical lines, the SD (number of animals: 1013). *Different from the CTR

group and #different from the pH group for each experimental period (P< 0.05).

(34.66.2%) or only to pH 5 for 24h (36.25.9%) and 96h(30.74.8%) was significantly greater than those of the respec-

tive control groups (30.75.6 and 24.53.9). The exposure to Alor only to acid pH for 6 h did not affect fish hematocrit, whichdid not differ from control value (21.83.2%) (Fig. 1B). The num-ber of red blood cells also increased significantly after 24 and

96 h of exposure to Al (29.1% and 27.8%, respectively) and to pH5 (21.2% and 27.2%, respectively), in relation to respective controls(Fig. 1C). After 6 h, only fish exposed to Al showed RBC counts sig-nificantly greater (48.3%) than respective control fish (Fig. 1C).

3.2. Plasma ions and osmolarity

Plasma osmolarity decreased significantly in fish exposed toAl for 24 h (6.8%) and 96 h (12.6%) in relation to respective con-

trols (Fig. 2A). Sodium plasmatic concentrations were lower thanrespective control values after both time points, decreasing from127.5 to 112.4 mM after 24 h and from 132.7 to 109.3mM after96 h of Al exposure (Fig. 2B). Plasma Cl levels of fish exposed to

Al for 24 and 96 h were also significantly lower than control fish(decreased 3.9% and 11.7%, respectively) (Fig. 2C). Plasma osmolar-ity as well as the plasmatic concentrations of sodium and chloride

offish exposed topH 5 didnot differ fromcontrol values throughoutthe study (Fig. 2AC). Plasma K+ concentrations showed large vari-ability among different exposure times and experimental groups(Fig. 2D).

3.3. Plasma protein, glucose and cortisol

Plasma protein levels of Al exposed fish were significantlygreater than in control fish after 6h (34.7%), 24h (24%) and 96h(364%). In fish exposed only to pH 5, for 6 and 24h, plasma protein

Fig. 4. Immunohistochemistry location of the Na+/K+ATPase enzyme in the chloride cells (CC) of P. lineatus used in the experiment of 96 h. The arrows indicate strong

immunoreactivity of Na+/K+ATPase in the fish of CTR group (A). In the gills of fish exposed to Al group (C), white arrows indicate CC weakly stained and also a small number

of CC, and in the fish exposed to pH group (B) is noted an increased number of lamellar and filamental CC. Scale bar: 30 m.


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Fig. 5. Density of chloride cells in the lamellar and filamental regions of the gills of

P.lineatus exposed to6, 24and 96h toCTR,pH orAl groups.*Differentfromthe total

number of CCin therespectiveCTR group; small lettersare related tothe lamellarCC

and capital letters are related to filamental CC. Different letters indicates statistical

difference for each parameter in each experimental period (P< 0.05).

levels were not affected, but increased significantly (352%) in rela-tion tocontrol groupafter96 h exposure(Fig.3A).FishexposedtoAlalso showed significantly higher levels of glucose after 6h (45.3%),

24 h (213%) and96 h (492%) than respective control groups(Fig.3B).Plasma glucose levels were not significantly different between pHand respective control groups, in any experimental period (Fig. 3B).Plasma cortisol concentration did not change significantly amongdifferent treatments, in any experimental period, ranging from 16

to 34ng mL1 (Fig. 3C).

3.4. CC distribution and density

P. lineatus from the control group has large number of CC dis-tributed throughout the filament and lamellar epithelium (Fig. 4A).

The exposure to acid water did not change such CC distribution(Fig. 4B), however, after Al exposure CC in the lamella disappearedand those in the filament were extremely reduced (Fig. 4C). Fig. 5shows the changes in CC density and localization in the gills of fish

from control, pH and Al groups. In general, exposure to acid waterinduced an increase in CC density in both, filament and lamella(also shown in Fig. 4B). Conversely, aluminium exposure, althoughin acid water, resulted in significant reduction of CC density in the

lamella after 6 h and in both filament and lamella after 24 and96h.

3.5. Gill Na+/K+ATPase activity

Na+/K+ATPase activity in the gills of fish exposed to Al,at all exposure times, showed significant inhibition (on aver-

age, 50% reduction) when compared to the animals fromcontrol groups (Fig. 6), which Na+/K+ATPase activity was1.22M Pi/mg proteinh1. Acid exposure did not produce any sig-

Fig. 6. Percentage of activity of Na+/K+ATPase enzyme in the gills ofP. lineatus

exposed to6, 24and 96h toCTR, pHor Al groups.*Different from theCTR group and#

different from the pH group for each experimental period (P< 0.05).

nificant difference in the Na+/K+ATPase activity in relation to CTRgroups (Fig. 6).

4. Discussion

The neotropical freshwater fish P. lineatus exposed to alu-minium, at low water pH (pH 5.0), exhibited osmoregulatorydisruptionindicatedby plasma Na+ andCl concentration decrease,probably due to the reduction of chloride cells density in the gills

and consequent reduced Na+/K+ATPase activity. Stress condition,supported by high glucose content in plasma, may have corrobo-rated to osmoregulatory disturbance.

The concentration of dissolved Al and the pH value used in this

study have already been reported in surface waters in Brazil ( Laraet al., 2001; Flues et al., 2002) due to natural causes or becauseof anthropogenic emissions. The concentration of 200g L1 ofdissolved Al corresponds to the maximal concentration allowed

by the Brazilian guidelines for freshwater. However, the results ofthe present study clearly showed that this concentration promotessome serious effects on fish osmoregulation.

Water pH of 5 is not lethal to P. lineatus (Takasusuki et al., 2004)

although it has been found to be the maximal tolerated for most

freshwater fishes (Playle and Wood, 1990; Polo, 1995; Waring andBrown, 1995). Values of pH between 6 and 9 are recommended forfreshwater used for the protection of fish communities in Brazil

(CONAMA 357, 2005). However, episodes of rapid acidification incontinental water bodies may occur during ecological accidents. Insuch situations Al from the soil is mobilised providing high eleva-tion of aluminium in its dissolved and more toxic form affecting

fish (Monette and McCormick, 2008).In the present study, the increased hematocrit and RBC counts

after 24 and 96h exposureto acid water and toAl in acid water can-notbe consideredas a good indicatorof Alexposure, assuggested by

Witters etal.(1996), at least,for P.lineatus. Changes in bloodparam-etersofsamespeciesexposedonlytolowwaterpH(pH4.5at20and30 C) have been described by Carvalho and Fernandes (2006). The

increased number of RBC and hemoglobin content may representthe secondary stress response, which leads to an increased RBC inthe circulation, because of spleen contraction, to improveO2 uptakefor metabolism (Brown, 1993; Wendelaar Bonga, 1997; Hontela,1998). Elevated blood parameters were also described in Salmoni-

formes fish, such as Oncorhynchus mykiss and Salmo trutta, and inthe neotropical fish Leporinusmacrocephalus afteracute exposuretoAl in acid water (Witters et al., 1990; Witters et al., 1996; Barcarolliand Martinez, 2004). In these works, Al concentrations variedfrom

15to200g L1. Polo andHytterd(2003) alsoregisteredelevatedblood parameters in salmons exposed to Al concentrations from 28to 359g L1 in alkaline waters.

Stress response in P. lineatus was also indicated by the increased

plasma glucose after 6 h of Al exposure. The elevation in plasma

glucose is a typical response for any animal facing a stressing situ-ation (Brown, 1993; Lohner et al., 2001) and it is mediated by thecatecholamines and cortisol release. The increase in plasma glucose

was the result of gluconeogenic processes or hepatic glucogenoly-sis to supply the increase in the energy demand caused by stress(Witters et al., 1996). Hyperglycaemia has been reported, by sev-eral authors, in fishes exposed to copper (Tavares-Dias et al., 2002),

to aluminium (Witters et al., 1996; Barcarolli and Martinez, 2004)and other different stressing situations (Mommsen et al., 1999).As the catecholamines are rapidly eliminated from the circulation,the maintenance of the high plasma glucose levels, as observed in

the present study, could have resulted from cortisol release, whichmight have occurred just after exposure to pollutant (Iwama etal., 2004). Cortisol is the main corticosteroid hormone in fish, and

toxicagents canthereby interfereon its dynamics (Mommsen et al.,


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1999). The absence of significant changes in plasma cortisol, in thepresent study, corroborates with the data previously reported by

LangianoandMartinez(2008)and Pereira-Maduenhoand Martinez(2008), for the same fish submitted to different stressful agent. Ingeneral, the increase in cortisol occurs between 0.5 and 1 h afterexposureto a contaminant(Barton, 2002) returningto thebasallev-

elsaftersome minutes or fewhours(Mommsen etal., 1999;Slomanetal.,2001; Iwama etal.,2004). The mobilisation of energy reservesas part of the stress response also includes protein metabolism(Heath, 1987; Adams et al., 1990; Mommsen et al., 1999). In the

case of the present study, the high values of total protein observedin the animals exposed to Al may be the result of two events: pro-tein mobilisation to meet the higher energy demand imposed bystress, or cell damage and proteins release due to direct action of

the metal on the cells (Exley et al., 1991; Wilson, 1996).Metals in water may act directly or indirectly via stress hor-

mones in the gills causing changes in osmo-ionic homeostasis.Freshwater fish undergo passive influx of water and ions efflux and

equilibrates the osmotic fluxexcreting large volume of diluted urineand taking actively ions by the gills (Evans et al., 2005; Lingwoodet al., 2006; Hwang and Lee, 2008). Aluminium, at concentrationsbetween 100 and 200g L1 and at a pH of near to 5.0, interactswith the gills and favour electrolytes loss (Dietrich and Schlatter,

1989; Exley et al., 1991). However, the present data suggest that Alinterfered on sodium uptake by the gills of P. lineatus. Reductionson plasma ions concentrations in fish exposed to Al were already

reported (Dietrich and Schlatter, 1989; Exley et al., 1991; Witterset al., 1996). Reductions in plasma sodium and chloride concen-trations were found in L. macrocephalus exposed to Al in acid pH(Barcarolli and Martinez, 2004) and salmon in similar conditions

(Monette and McCormick, 2008).Decreased plasma ionicconcentrations in stressed fishes cannot

be related only to the reduced active uptake of ions, the increasein paracellular permeability of the branchial epithelium, which

increases the passive efflux of ions, might represent another cause(Monette and McCormick, 2008). In this study, the analysis of chlo-ride cells (CC) and of Na+/K+ATPase was used as an approach to

understand which stage of the osmoregulation process would bedamaged in fish exposed to Al.

Na+/K+ATPase is a protein that is linked to the cell membraneand it uses energy from the hydrolysis of ATP in order to transport2K+ into the cell and 3Na+ out from the cell to the blood, being of

great importance in the gills of teleosts (Lingwood et al., 2006; Silvaet al., 2007; Hwang and Lee, 2008). There is a positive correlationbetween the staining of the CC and the activity of this enzyme inthe branchial epithelium of fish (Dang et al., 2000). In the present

study, the lower activity of Na+/K+ATPase determined in the gillsof the fish exposed to Al could be related to the lower activity ofthis enzyme in the poorly stained CC and to the smaller number ofCC found in the filaments (due to the death of these cells through

apoptosis and/or necrosis). Monette and McCormick (2008) also

observed similar results in young salmon after acute exposure toAl in acid pH. These authors claim that the CCs are the main sitefor the accumulation of aluminium in the gills, and consequently

the death of these cells would facilitate the elimination of Al fromthis organ. The few CC noted in the fish exposed to Al seemed tobe displaced mostly to the lamellar region rather than in the fil-ament. Dang et al. (2000) obtained similar results in Oreochromis

mossambicus exposed to copper, i.e., both a decreased number of CCin the filaments and the migration of CC to the branchial lamellae.CC in gill lamellae would be closer to the bloodstream, facilitatingion uptake, which can also mean that these cells are more resistant

to the metal than the CC that remain in the filament (Dang et al.,2000). Some of theCC that were found in the lamellae could repre-sent immature cells as well, with a smaller quantity and/or activity

of Na+

/K+

ATPase, and therefore, they were less stained. The pres-

ence of these immature cells might representthe action of cortisol,which interferes with CC differentiation. In the present study, this

idea would be supported by the occurrence of secondary stressresponses (such as the increase in blood glucose and hematologicalparameters). Besides, it is importantto point outthat the immatureCC couldshowgreater concentrations of metallothioneins, proteins

that could bind metals and protect thetissue from thedirect actionof metal ions (Dang et al., 2000).

The increase in the number of CC in fish exposed only to acidwater, at all experimental periods may be related to the mainte-

nance of acidbase balance (Clairborne et al., 2002; Sakuragui etal., 2003; Hwang and Lee, 2008), rather than directly related to ionuptake in these fish. Takasusuki et al. (2004) established a high tol-erance for changes in water pH for P. lineatus, however, a pH of 4.5

is more stressful to this fish species than one of 8.0.Until now, the exact role of CCs in the transport of Cl and Na+

across the gills and in the acidbase regulation is not well estab-lished. The exchange of HCO3

for Cl together with theH+/ATPase

to eliminateH+ creates an electrical potential that favoursthe influxof Na+ (Hwang and Lee, 2008). This may explain, at least in part,the increasing of these cells in P. lineatus exposed to low water pH,allowing fish to maintain an efficient active excretion of H+ and ion

regulation. H+/ATPase has been found in both theCC and pavement

cells of the gill (Goss et al., 1998;Clairborne et al., 2002; Hwang andLee, 2008).

In summary, the present study points out relevant results of

the toxicity of Al in acid water to a neotropical fish species, show-ing that P. lineatus experienced osmoregulatory disturbances. Thecauses of ionic unbalance is probably related to the effects of Al onNa+/K+ATPase activity, on the distribution andnumber of chloride

cells in the gills as well as the effects associated with the stressresponse caused by the presence of the metal.

Acknowledgments

The authors thank the Hatchery Station of State University of

Londrina (EPUEL) for the supply of fish and Helen Sadauskas Hen-rique and Marcelo Gustavo Paulino (UFSCar) for assistance duringthe immunohistochemistry assay. The Brazilian Council for Scien-

tific and Technological Development (CNPq) supported this work(grant no. 477073/2006-9). This work is part of the PhD The-sis of M.M.P.Camargo who received a doctoral scholarship fromCTHidro/CNPq. M.N.Fernandes and C.B.R. Martinez areresearchfel-

lows from CNPq and members of the Brazilian Institute of AquaticToxicology (INCT-TA, CNPq: 573949/2008-5).

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